The present invention relates to an improved process for the isolation and purification of Paclitaxel from natural sources.
U.S. Pat. No. 6,452,024 and its foreign counterparts all in the name of the present Applicant, CHAICHEM PHARMACEUTICALS INTERNATIONAL, disclose and claim a process of the above mentioned type, which is particularly interesting as compared to all the previous processes disclosed as xe2x80x9cprior art in the preamble of this U.S. patent. More specifically, the process disclosed in this U.S. patent is interesting inasmuch as it permits:
to make easier the obtention of a biomass after extraction of the barks, needles and/or branches of Taxus of different species;
to increase the amount of biomass which is so obtained and has to be purified by chromatography;
to reduce the steps of purification;
to increase the amount of obtained Paclitaxel; and
to reduce the production cost to a more economical level.
The process disclosed in this U.S. Pat. No. 6,452,024 basically comprises the following steps:
a) extracting a raw material comprising Paclitaxel from a natural source of taxanes with an organic solvent;
b) contacting the raw material with a basic medium or an acidic medium to obtain a biomass by precipitation, isolating and drying said biomass;
c) removing resin and natural pigments from the isolated and dried biomass by dissolving the biomass in acetone and then adding to it at least one non-polar solvent such as hexane or heptane, until a Paclitaxel-enriched oily phase is obtained;
d) contacting the Paclitaxel-enriched oily phase recovered in the preceding step with an acidic medium when step (b) was carried out with a basic medium, or with a basic medium when step (b) was carried out with an acidic medium, in order to obtain a precipitate by precipitation, isolating the precipitate and drying it; and
e) chromatographically purifying at least once a solution of the isolated precipitate in a volatile solvent, and crystallizing at least once the purified solution obtained by chromatography.
In the above U.S. patent, it is also disclosed that step (e) preferably comprises:
e1) a first chromatographic purification comprising dissolving the precipitate isolated ion step (d) in a volatile solvent, preparing a mixture of the so-obtained solution with silica gel, treating the mixture in a chromatographic column containing silica gel, and recovering Paclitaxel-enriched fractions;
e2) a second chromatographic purification comprising by evaporating to dryness the Paclitaxel-enriched fractions recovered in the preceding step until a residue is obtained, and preparing a mixture by solubilizing said residue in a volatile solvent, the mixture being repurified by chromatography under the same conditions as in the preceding sub-step in order to obtain other Paclitaxel-enriched fractions;
e3) a first crystallization comprising evaporating to dryness the other Paclitaxel-enriched fractions obtained in the preceding sub-step until a residue is obtained, preparing a mixture of this residue in acetone and crystallizing the Paclitaxel contained in the mixture with a non-polar solvent;
e4) a second crystallization comprising solubilizing in acetone Paclitaxel crystals obtained in the preceding sub-step and recrystallizing the Paclitaxel under the same conditions as in the preceding sub-step;
e5) a third chromatography purification comprising solubilizing the crystals obtained by recrystallization in the preceding sub-step in a volatile solvent to obtain a solution, preparing a mixture of this solution with silica gel and treating said mixture in a chromatographic column containing silica gel in order to obtain, with an elution solvent, further Paclitaxel-enriched fractions; and
e6) a third crystallization comprising evaporating to dryness the further Paclitaxel-enriched fractions obtained in the preceding sub-step until a residue is obtained, solubilizing the residue in an alcohol, cetone or an alcohol-cetone mixture to obtain another mixture, and crystallizing the Paclitaxel contained in the other mixture with water.
As aforesaid, the process disclosed in the above U.S. Pat. No. 6,452,024 is very interesting inasmuch as it is much simpler, efficient and cost-effective than the existing processes. However, this process still calls for numerous steps of separation by chromatography and purification by crystallization that are necessary to remove the large quantities of solvent-soluble impurities that are obtained when the extraction is carried out using needles and twigs from natural sources of taxanes. This leads to a still high production cost due in particular to the low amount of Paclitaxel in the different species of Taxus. Moreover, the amount of biomass which can be purified, is very limited because of the small sizes of the chromatography columns and because of the low yield in Paclitaxel obtained after purification.
The object of the present invention is to provide an improved process which solves most of the problems mentioned hereinabove.
More particularly, the object of the invention is to provide an improved process which permits:
to obtain a solution which contains the most of Paclitaxel and analogs after extraction of the barks, needles and/or branches of Taxus of different species;
to reduce the impurities and the volume of the Paclitaxel and analogs extract with which the biomass is obtained by precipitation;
to further increase the amount of biomass which has to be purified by chromatography;
to further reduce the steps of purification;
to further increase the amount of obtaining Paclitaxel; and finally
to further reduce the production cost to a more economical level.
The improved process according to the invention for the extraction and purification of. Paclitaxel from a natural source of taxanes containing Paclitaxel, comprises the following basic steps:
a) washing with deionized or pure water, a raw material comprising Paclitaxel, said raw material coming from said natural source of taxanes, such a washing allowing removal of soluble impurities from said raw material;
b) extracting with an organic solvent from said washed raw material, a wet raw material comprising Paclitaxel;
c) contacting said wet raw material with a salt to obtain a biomass by precipitation, and isolating and drying said biomass;
d) removing resin and natural pigments from the so isolated and dried biomass by dissolving said biomass in acetone or an acetone-hexane mixture and then adding thereto at least one-polar solvent until a Paclitaxel-enriched oil phase is obtained;
e) purifying by chromatography at least once the Paclitaxel-enriched oily phase obtained in the preceding step in a volatile solvent to obtain a purified solution and crystallization at least once the purified solution obtained by chromatography.
The crystallized product that is so obtained is actually a mixture of Paclitaxel crystals, which, after filtration and drying of the crystals, basically consists of:
about 60% of crystals having a purity higher than 99%;
about 30% of crystals having a purity higher than 98% ( less than 99%); and
about 10% of crystals having a purity higher than 92% ( less than 98%).
The crystals having purities less than 99% can be separated, mixed together and subjected to subsequent purification by chromatography in order to get more of the final product having a purity higher than 99%.